Recently, organ transplantation has widely been carried out in many countries. Development of highly effective immunosuppressants (e.g., Cyclosporin and FK506) has solved the problem of rejection of organs transplanted from man to man, however, lack of donors has become a serious problem. Such a problem has prompted studies on animal-to-man organ transplantation, namely xenotransplantation. Although approximately 3,500 instances of heart transplantation have been performed annually in European countries and the United States, they cover only 20 to 30% of patients who need heart transplantation. Use of animals closely related to human beings as donors (for example, such primates as baboons and chimpanzees) involves a great deal of difficulty due to shortage of these animals and their high intelligence, but use of domestic animals as donors involves less problems. Particularly, pigs have advantages of easy supply due to mass rearing, their organ sizes similar to those of man, and established basic technology including maintenance of the strains. Consequently, organ transplantation from pig to man has been studied.
Of rejections occurring in pig-to-man organ transplantation, acute rejection by major histocompatibility complex (MHC)-related cellular immunity may not occur, since evolutional relatedness between pigs and man is so scarce that there is no similarity between their MHCs. Moreover, application of the effective immunosuppressants may avoid such rejection, if ever occurs.
Human blood, however, contains endogenous antibodies against pigs (namely, natural antibodies). Consequently, if a porcine organ is transplanted to man, the natural antibodies recognize the organ (antigen) resulting in formation of antigen-antibody complexes, which activate human complements. The activated human complements cause necrosis of the transplanted organ (rejection). Such a phenomenon occurs immediately (within an hour) after transplantation, so it is termed hyperacute rejection.
No drug preventing hyperacute rejection caused by complement activation has ever been developed. No human organ is injured by human complements, since factors preventing complement activation are expressed in human organs. Such factors are named complement inhibitors (or complement-inhibiting factors). Of the complement inhibitors, three factors, DAF (decay accelerating factor, CD55), MCP (membrane cofactor protein, CD46) and CD59, are important. It is believed that DAF and MCP inhibit activation of complements by accelerating the destruction of C3b and C3/C5 convertase, and CD59 does so by inhibiting the C9 step.
The complement inhibitors are species specific. Porcine complement inhibitors can inhibit the complement activity of pigs but not that of man. The porcine complement inhibitors cannot inhibit human complements activated by the porcine organ transplanted to man. Therefore, the porcine organ transplanted to man undergoes necrosis.
Pig-to-man organ transplantation triggers not only hyperacute rejection but also thrombin formation and platelet coagulation, resulting in thrombosis in the hosts vascular system as well as the transplanted organ. Components of the blood coagulation pathway, such as thrombin, fibrin and fibrin degradation products may also amplify tissue damage, modify immune responses and augment inflammatory responses (Xenotransplantation, vol. 3(1). 24-34, 1996).
Such problems arising when a porcine organ is transplanted to man will be solved, if human complement inhibitors and/or such thrombosis-inhibiting factors as thrombomodulin (collectively termed as complement inhibitors in the following) are expressed in the porcine organs by genetic engineering. In transplantation of the porcine heart, there will be no problem if the human complement inhibitors are being expressed by porcine vascular endothelial cells.
From such a viewpoint, studies on recombinant pigs (transgenic pigs) integrated with human complement-inhibitor genes have widely been carried out.
It has also been considered to be useful to express such factors that are capable of suppressing thrombosis (e.g., thrombomodulin, Proceedings of the XVth World Congress of the Transplantation Society, pp. 77-79, 1995) with or without the human complement inhibitors in the porcine organs by genetic engineering.
As described above, xenotransplantation by using transgenic pigs integrated with the human complement-inhibitor genes have been studied. Up to the present, promoters derived from the human complement-inhibitor gene or viruses have been used to prepare such transgenic pigs. For the complement inhibitors to be expressed in pigs, however, the promoters originating from pigs may be more efficient. To obtain such promoters, cDNA of the porcine complement inhibitors is needed. Therefore, the present inventors carried out studies to isolate and purify cDNA encoding a porcine complement inhibitor (termed pMCP in the following) and succeeded in isolating and sequencing its cDNA (see Japanese Pat. Appln. No. 178254/1995).
The present inventors further studied and succeeded in identifying and sequencing the promoter region of PMCP by preparing porcine genomic libraries and then screening them with pMCP's cDNA as a probe.
As described above, pMCP's promoter of the invention was derived from pMCP's genomic DNA, which was isolated by using cDNA of pMCP. pMCP's cDNA was derived from RNA transcribed in porcine vascular endothelial cells. Namely, pMCP's promoter of the invention regulates expression of pMCP in the porcine vascular endothelial cells. Consequently, by using pMCP's promoter of the invention, genes of human complement inhibitors and those of such thrombosis-inhibiting factors as thrombomodulin can be expressed in the porcine organs, particularly the porcine vascular endothelial cells. Furthermore, by using pMCP's promoter of the invention, various structural genes can effectively, selectively and specifically be expressed in the porcine organs, particularly the porcine vascular endothelial cells.
On the other hand, the promoters derived from the human complement-inhibitor gene or viral genome, all of which had been employed in the previous studies, could neither selectively, specifically nor effectively express the human complement inhibitor in the porcine endothelial cells.
This invention was accomplished on the basis of such findings. The purpose of the invention was to provide DNA possessing an activity of pMCP's promoter.